CN109759150A - Controllable extraining sampling device, sample injection method and application based on micro- free stream cataphoresis - Google Patents
Controllable extraining sampling device, sample injection method and application based on micro- free stream cataphoresis Download PDFInfo
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- CN109759150A CN109759150A CN201910092648.9A CN201910092648A CN109759150A CN 109759150 A CN109759150 A CN 109759150A CN 201910092648 A CN201910092648 A CN 201910092648A CN 109759150 A CN109759150 A CN 109759150A
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Abstract
The present invention relates to controllable extraining sampling device, sample injection method and applications based on micro- free stream cataphoresis.The controllable extraining sampling device, including micro- free stream cataphoresis chip, micro- free stream cataphoresis chip includes split tunnel, and electrode is arranged in the two sides of the split tunnel；Sample introduction zone, the inflow end of the split tunnel is arranged in the sample introduction zone, and the sample introduction zone is provided with a sample channel and two buffer channels, the sample channel is perpendicular to two buffer channels, and two buffer channels make the sample introduction zone in " ┴ " type on the same axis；The outflow end of split tunnel is arranged in exit passageway, the exit passageway.Controllable extraining sampling technology of the present invention is separated suitable for continuous free stream cataphoresis, can be effectively reduced the initial bandwidth of sample, be improved separating property, and chip manufacturing requires, is at low cost.
Description
Technical field
The present invention relates to micro- free stream cataphoresis technical fields, and in particular to the controllable extraining sampling based on micro- free stream cataphoresis
Device, sample injection method and application.
Background technique
Micro- free stream cataphoresis (Micro free-flow electrophoresis, μ FFE) is a kind of continuous high speed electricity point
From micromation analysis method.It has a wide range of application, and the separation of even cell is pure from small molecule fluorescent dyestuff to large biological molecule
Change all has been reported that.Compared with commercialized large-scale free stream cataphoresis, micro- free stream cataphoresis has quick, portable, sample and solvent
The advantages that consumption is small.Also it can access on-line system, become micro- preparative separation analysis in continuous sample preparation or multistep synthesis
Committed step.The development of current micro- free stream cataphoresis is largely determined by complicated device processing and the lower separation property of controllability
Energy.
As Capillary Electrophoresis, the separating degree of free stream cataphoresis is by electric field strength, sample electromobility, sample in electric field
In residence time and sample broadening determine.Unlike Capillary Electrophoresis in free stream cataphoresis sample flow in disengagement chamber
It deflects, generate additional sample broadening, seriously affected separating effect.In order to improve the separating effect of free stream cataphoresis, adjust
Field intensity of economizing on electricity is the most frequently used and direct method, but excessive electric field strength can cause the raising of Joule heat, destroy separation.
Using micro-processing technology, the free stream cataphoresis realized in microchannel can radiate rapidly, effectively reduce Joule heat.Another kind improves
The method of free stream cataphoresis separating effect is control sample broadening.The sample of free stream cataphoresis broadens mainly by initial bandwidth, expansion
It dissipates broadening, fluid dynamics broadening and sample flow and deflects four combined factors influences.In recent years, there are a large amount of computer simulations
And laboratory research, the method that design reduces fluid dynamics broadening and reduces diffusion broadening, it was also proposed that inclined for sample flow
The novel separating degree analysis method turned.However, as diffusion broadening, fluid dynamics broadening and sample flow deflection further subtract
Small, initial bandwidth is increasingly becoming the leading factor for influencing sample broadening and separating property.But in current research at the beginning of sample
Beginning bandwidth is often assumed to be constant, studies less.Based on the above analysis, there is a need in the art for propositions can be with Effective Regulation sample
The initial bandwidth of product, the novel sampling technique for realizing quick separating.
Summary of the invention
It is an object of the invention to the deficiencies for the bandwidth research initial for sample of current micro- free stream cataphoresis, provide
A kind of controllable extraining sampling device, sample injection method and its application, the technology separate suitable for continuous free stream cataphoresis, can have
Effect ground reduces the initial bandwidth of sample, improves separative efficiency, and chip manufacturing requires, is at low cost.Effectively to solve in the prior art
Deficiency.
In order to achieve the above objectives, the present invention adopts the following technical scheme:
Controllable extraining sampling device based on micro- free stream cataphoresis, including micro- free stream cataphoresis chip, micro- free flow
Electrophoresis chip includes split tunnel, and electrode is arranged in the two sides of the split tunnel；Sample introduction zone, the sample introduction zone are arranged at described point
Inflow end from channel, and the sample introduction zone is provided with a sample channel and two buffer channels, the sample channel hangs down
Directly in two buffer channels, and two buffer channels make the sample introduction zone in " ┴ " type on the same axis；Outlet is logical
The outflow end of split tunnel is arranged in road, the exit passageway.
Further, the sample channel width: buffer channel width: split tunnel width is 1:1-5:1-10；Institute
Stating split tunnel width is 0.6-6.0mm, and length 2.5-25mm, depth is 20-100 μm.
Further, micro- free stream cataphoresis chip material is cyclic olefine copolymer (COC), glass, poly dimethyl silicon
Any one in oxygen alkane (PDMS) or polymethyl methacrylate (PMMA), the electrode material are platinum filament, spun gold, carbon nanometer
Any one in pipe.
Further, the exit passageway has two and from split tunnel outflow end to two sides horizontal extension, so that described
Micro- free stream cataphoresis chip is integrally in " soil " type.
Micro- free stream cataphoresis chip of the invention is respectively arranged with buffer channel and sample channel, to realize not cocurrent flow
Measure the extraining sampling of ratio.In the case where not changing the conditions such as microchannel size, operation voltage, by simply regulating and controlling buffer
The regulation to sample separating property is mixed in Disengagement zone may be implemented with the flow-rate ratio of sample.Controllable folder of the present invention flows into
Sample technology is separated suitable for continuous free stream cataphoresis, can be effectively reduced the initial bandwidth of sample, be improved separating property, and chip
Production requires, is at low cost.
The controllable extraining sampling method based on micro- free stream cataphoresis that the present invention also provides a kind of, includes the following steps, 1), it is real
Before testing, sample introduction first is carried out full of replacement sample solution after whole device using buffer；2) electric field strength, is adjusted, then is adjusted slow
The ratio r of fliud flushing flow and sample flow, and photograph to record the initial bandwidth figure of sample and separation figure；
3) separation figure, is photographed to record in split tunnel downstream or detection is sampled to the effluent of exit passageway.
Further, in the step 2), the initial ratio r of control buffering flow quantity and sample flow is 1, is then increased again
The ratio r of big buffering flow quantity and sample flow, and the initial bandwidth figure of sample and separation figure are photographed to record respectively.
Further, the modification scope of the electric field strength is 10-500V/cm；The buffering flow quantity and sample flow
Ratio adjustable range be 1-256.Guaranteeing at this time can sample bandwidth regulation to the greatest extent under sampling condition.
A kind of controllable extraining sampling device based on micro- free stream cataphoresis of the invention can obtain in fluorescent dye separation
Using.
A kind of controllable extraining sampling device based on micro- free stream cataphoresis of the invention can be applied in separation for amino acids.
A kind of controllable extraining sampling device and its sample injection method based on micro- free stream cataphoresis of the invention is suitable for continuous
Free stream cataphoresis separation.By controlling the ratio by buffering flow quantity and sample flow, sample can be reduced from injection port
Initial bandwidth is conducive to sample and realizes quick separating, while reducing sample introduction to reduce the broadening of the sample in entire separation process
The production of area channel requires, and reduces the chip manufacturing cost of micro- free stream cataphoresis.
In order to better understand and implement, the invention will now be described in detail with reference to the accompanying drawings.
Detailed description of the invention
Fig. 1 is the structural schematic diagram of micro- free stream cataphoresis chip of the controllable extraining sampling device of the present invention；
Fig. 2 is buffering flow quantity figure compared with sample initial bandwidth when sample flow ratio r is from 1 to 256；
Fig. 3 is the different sample figures compared with buffer flow-rate ratio is to fluorescent dye separating effect of the embodiment of the present invention 1；
Fig. 4 is the different sample figures compared with buffer flow-rate ratio is to separation for amino acids effect of the embodiment of the present invention 2.
In figure, 1, split tunnel；2, sample channel；3, buffer channel；4, exit passageway；5, electrode；A, sample introduction zone；b,
Disengagement zone；C, it takes pictures detection zone.
Specific embodiment
Below with reference to embodiment, present invention is further described in detail
As shown in FIG. 1, FIG. 1 is the structural schematic diagram of micro- free stream cataphoresis chip of extraining sampling device of the invention controllable,
The present invention provides the controllable extraining sampling device based on micro- free stream cataphoresis, including micro- free stream cataphoresis chip, the device be
It is realized on cyclic olefine copolymer (COC) chip, or glass, dimethyl silicone polymer (PDMS) or polymethylacrylic acid
Any one in methyl esters (PMMA) chip.Micro- free stream cataphoresis chip includes that split tunnel 1, sample introduction zone a and outlet are logical
The inflow end of the split tunnel 1 is arranged in road 4, the sample introduction zone a, and the outflow of split tunnel 1 is arranged in the exit passageway 4
End.
The two sides of the split tunnel 1 are arranged electrode 5, the electrode 5 be inlayed from platinum filament and with hot pressing form, electrode
5 points are positive and negative anodes, and external power supply (not shown) forms electric field.1 width of split tunnel is 0.6-6.0mm, and length is
2.5-25mm, depth are 20-100 μm；Preferably, 1 width of split tunnel is 1.6mm, and length 5mm, depth is 70 μm.It is described
Sample channel width: buffer channel width: split tunnel width is 1:1-5:1-10, it is furthermore preferred that the sample channel 2 is wide
Degree: 3 width of buffer channel: 1 width of split tunnel is 1:1:3.
The sample introduction zone a is provided with a sample channel 2 and two buffer channels 3, and the sample channel 2 is perpendicular to two
Buffer channel 3, and two buffer channels 3 make the sample introduction zone a in " ┴ " type on same level axis.Sample is logical
It converges with two buffer channels 3 and is communicated with split tunnel 1 in road 2.It further include syringe pump (not shown), the syringe pump
It is separately connected buffer channel 3 and sample channel 2；The buffer channel 3 on the both sides " ┴ " type sample introduction zone a and a binary channels are injected
Pump is connected, and intermediate sample channel 2 passes through a single syringe pump load sample.Sample and buffer may be implemented by syringe pump
Enter split tunnel 1 by pressing from both sides stream mode, syringe pump can control the flow of liquid, buffer so as to control by syringe pump
Liquid and sample flow ratio.
The exit passageway 4 is from 1 outflow end of split tunnel to two sides horizontal extension, two exit passageways 4 and two bufferings
Liquid channel 3 is parallel to each other, and than two buffer channels 3 are longer in the horizontal direction for two exit passageways 4.In this way, make
Obtaining micro- free stream cataphoresis chip is integrally in " soil " type.
Controllable extraining sampling device based on micro- free stream cataphoresis of the invention, sample solution and buffer are using folder stream side
Formula enters split tunnel 1, and under electric field action, in Disengagement zone, b is separated, can be simple by syringe pump during this
Regulate and control the flow-rate ratio of buffer and sample and then changes the initial bandwidth of sample.It is rectangular on the ground that sample introduction zone a and Disengagement zone b cross
Heterodyne shines detection zone c, and detection zone c for recording the initial bandwidth of sample in real time.As shown in Fig. 2, buffering flow quantity and sample
The initial bandwidth of sample compares figure when flow ratio r is from 1 to 256.
Of the invention also provides the controllable extraining sampling method based on micro- free stream cataphoresis, includes the following steps,
1) before, testing, sample introduction first is carried out full of replacement sample solution after whole device using buffer；
2) electric field strength, is adjusted, then adjusts the ratio r of buffering flow quantity and sample flow, and the detection zone c that takes pictures is carried out
Photograph to record the initial bandwidth figure of sample；
3) separation figure, is photographed to record in split tunnel downstream or detection is sampled to the effluent of exit passageway, and
The same position of split tunnel reads fluorescence intensity and makees fluorescence intensity figure in figure.
In the step 2), the initial ratio r of control buffering flow quantity and sample flow is 1, then increases buffer again
The ratio r of flow and sample flow, and the initial bandwidth figure of sample and separation figure are photographed to record respectively.
The modification scope of the electric field strength is 10-500V/cm；The ratio of the buffering flow quantity and sample flow is adjusted
Range is 1-256.Guaranteeing at this time can sample bandwidth regulation to the greatest extent under sampling condition.
Controllable extraining sampling device and sample injection method through the invention are not changing microchannel size, operation voltage etc.
In the case where condition, the flow-rate ratio by simply regulating and controlling buffer and sample is realized and then can change the initial bandwidth of sample
Regulation to sample separating property is mixed in the b of Disengagement zone.Controllable extraining sampling device based on micro- free stream cataphoresis of the invention
It can be separated in fluorescent dye and obtain applications well in separation for amino acids.
Illustrate the controllable extraining sampling device of the invention based on micro- free stream cataphoresis, side below by two embodiments
Method and application.
Embodiment 1: the separation of fluorescent dye
This example uses the 1mmol/L HEPES containing 0.05% hydroxypropyl methyl cellulose (w/w) as buffer, uses
Buffer concentration is 500 μm of ol/L rhodamine Bs and rhodamine 6G mixed solution as sample solution.Before experiment, first make
Sample introduction is carried out full of replacement sample solution behind entire channel with buffer.When experiment, control sample solution flow is 20 μ L/min,
Buffering flow quantity is 20 μ L/min, and sample solution and buffer volume flow ratio r are 1.After the system stabilizes, adjust voltage to
20V photographs to record phenomenon.In the case where voltage keeps 20V constant, adjusting sample solution flow to 0.156 μ L/min, at this time
R is 128, photographs to record phenomenon.
When sample solution and buffer flow-rate ratio r increase to 128 from 1, the initial bandwidth of sample narrows down to 35 μ from 800 μm
m.Fluorescence intensity is read in split tunnel downstream same position and is mapped.Separating effect such as Fig. 3 of the fluorescent dye mixing sample
It is shown.
Embodiment 2: the separation of amino acid
Amino acid derived method: 10mmol/L NaHCO is used3Solution prepares 25mmol/L amino acid solution respectively, prepares
4mg/mL NBD-F methanol solution.It is molten that 600 μ L 4mg/mL NBD-F methanol are added into 1.2mL 25mmol/L amino acid solution
Liquid reacts 15min under 80 DEG C of water-baths.
This example uses the 1mmol/L HEPES containing 0.05% hydroxypropyl methyl cellulose (w/w) as buffer, uses
Buffer concentration is respectively the glutamic acid (Glu) of 2.25mmol/LNBD-F label and relying for 1.5mmol/L NBD-F label
Propylhomoserin (Lys) mixed solution is as sample solution.Before experiment, first using buffer full of replacement sample solution behind entire channel into
Traveling sample.When experiment, control sample solution flow is 20 μ L/min, and buffering flow quantity is 20 μ L/min, sample solution and buffering
Flow quantity ratio r is 1.After the system stabilizes, voltage is adjusted to 34V, photographs to record phenomenon.In the situation that voltage keeps 34V constant
Under, sample solution flow is adjusted to 0.625 μ L/min, and r is 32 at this time, photographs to record phenomenon.
When sample solution and buffer flow-rate ratio r increase to 32 from 1, the initial bandwidth of sample narrows down to 56 μm from 890 μm.
Fluorescence intensity is read in split tunnel downstream same position and is mapped, and the separating effect of the amino acid aggregate sample is as shown in Figure 4.
The embodiments described above only express several embodiments of the present invention, and the description thereof is more specific and detailed, but simultaneously
It cannot therefore be construed as limiting the scope of the patent.It should be pointed out that coming for those of ordinary skill in the art
It says, without departing from the inventive concept of the premise, various modifications and improvements can be made, these belong to protection of the invention
Range.
Claims (9)
1. the controllable extraining sampling device based on micro- free stream cataphoresis, including micro- free stream cataphoresis chip, it is characterised in that: described
Micro- free stream cataphoresis chip includes split tunnel, and electrode is arranged in the two sides of the split tunnel；
Sample introduction zone, the inflow end of the split tunnel is arranged in the sample introduction zone, and the sample introduction zone is provided with a sample and leads to
Road and two buffer channels, the sample channel is perpendicular to two buffer channels, and two buffer channels are in same axis
Make the sample introduction zone in " ┴ " type on line；
The outflow end of split tunnel is arranged in exit passageway, the exit passageway.
2. the controllable extraining sampling device according to claim 1 based on micro- free stream cataphoresis, it is characterised in that: the sample
Product channel width: buffer channel width: the ratio of split tunnel width is 1:1-5:1-10；The split tunnel width is
0.6-6.0mm, length 2.5-25mm, depth are 20-100 μm.
3. the controllable extraining sampling device according to claim 1 based on micro- free stream cataphoresis, it is characterised in that: described micro-
Free stream cataphoresis chip material is cyclic olefine copolymer (COC), glass, dimethyl silicone polymer (PDMS) or polymethylacrylic acid
Any one in methyl esters (PMMA)；The electrode material is platinum filament, spun gold, any one in carbon nanotube.
4. the controllable extraining sampling device according to claim 1 based on micro- free stream cataphoresis, it is characterised in that: it is described go out
Mouth channel has two and from split tunnel outflow end to two sides horizontal extension, so that micro- free stream cataphoresis chip is integrally in
" soil " type.
5. the controllable extraining sampling method based on micro- free stream cataphoresis, it is characterised in that: include the following steps,
1) before, testing, sample introduction first is carried out full of replacement sample solution after whole device using buffer；
2) electric field strength, is adjusted, then adjusts the ratio r of buffering flow quantity and sample flow, and photograph to record the initial bandwidth of sample
Figure and separation figure；
3) separation figure, is photographed to record in split tunnel downstream or detection is sampled to the effluent of exit passageway.
6. the controllable extraining sampling method according to claim 5 based on micro- free stream cataphoresis, it is characterised in that: the step
It is rapid 2) in, the initial ratio r of control buffering flow quantity and sample flow is 1, then increases buffering flow quantity and sample flow again
Ratio r, and photograph to record the initial bandwidth figure of sample and separation figure respectively.
7. the controllable extraining sampling method according to claim 5 based on micro- free stream cataphoresis, it is characterised in that: the electricity
The modification scope of field intensity is 10-500V/cm；The ratio adjustable range of the buffering flow quantity and sample flow is 1-256.
8. the controllable extraining sampling device according to any one of claims 1 to 4 based on micro- free stream cataphoresis is in fluorescence
It is applied in dye separation.
9. the controllable extraining sampling device according to any one of claims 1 to 4 based on micro- free stream cataphoresis is in amino
It is applied in acid separation.
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